Bottom Line:
This silencing response is a rapid primary event distinct from repression mechanisms known to operate at later stages of DC maturation.The repressed genes function in pivotal processes--including antigen-presentation, extracellular signal detection, intracellular signal transduction and lipid-mediator biosynthesis--underscoring the central contribution of the silencing mechanism to rapid reshaping of DC function.Interestingly, promoters of the repressed genes exhibit a surprisingly high frequency of PU.1-occupied sites, suggesting a novel role for this lineage-specific transcription factor in marking genes poised for inducible repression.

Figure 5: Characterization of nascent-transcript-sequencing data. (A) The dot plots show a global analysis of altered nascent-transcript (left) and mRNA (right) expression induced in 1 h-LPS-treated Mo-DCs: results are represented as RPKM (reads per kb per million) on a log scale; induced (>2x), silenced (>2x) and unchanged genes are represented as green, red and black dots, respectively. (B) The dot plot compares changes in nascent-transcript and mRNA expression induced in 1 h-LPS-treated Mo-DCs: results are represented as fold change on a log scale; genes that are induced (>2x), silenced (>2x) and unchanged at the nascent-RNA level are represented as green, red and black dots, respectively. (C) The pie chart shows the percentage of deacetylated genes expressed more than 1 RPKM in immature Mo-DCs. The dot plot shows 1 h-LPS-induced changes in nascent-transcript (red dots) and mRNA (black dots) expression for deacetylated genes; genes are ordered with respect to their change in nascent-transcript expression; positions of representative genes are highlighted. (D) TFBS enrichment analyses were performed for promoters of genes that are induced (>2, 3 or 5-fold), deacetylated and silenced (>2, 3 or 5-fold), or exhibit no change (nc) in expression in Mo-DCs after 1 h of LPS treatment. TFBSs were defined according to JASPAR. The heat map shows the relative enrichment (z-score) of TFBSs that are significantly over-represented (P-value < 10−3). (E) The graphs summarize the z-scores and P-values for all TFBSs in genes that are induced >3-fold (top) or deacetylated and silenced >3-fold (bottom): NF-κB (green) and ETS (red) TFBSs are highlighted.

Mentions:
Examinations of selected genes indicated that nascent-transcript sequencing allows reliable quantification of transcription rates at genes that are silenced or induced in 1 h-LPS-treated Mo-DCs, including protein-coding and microRNA genes (Figure 4B, C and Supplementary Figure S4C). Global analyses indicated that markedly more changes in expression were evident at the primary-transcript level than at that of mRNA-abundance (Figure 5A and Supplementary Table S1). Significantly more genes were down-regulated at the nascent transcript level than at the mRNA level (Figure 5B and Supplementary Table S1), suggesting that most reductions in transcription rate induced by 1 h of LPS stimulation do not yet have a major impact on mRNA abundance. These results demonstrate that assessing gene silencing by nascent-transcript sequencing is significantly more reliable and has strongly improved temporal resolution compared to mRNA-profiling.

Figure 5: Characterization of nascent-transcript-sequencing data. (A) The dot plots show a global analysis of altered nascent-transcript (left) and mRNA (right) expression induced in 1 h-LPS-treated Mo-DCs: results are represented as RPKM (reads per kb per million) on a log scale; induced (>2x), silenced (>2x) and unchanged genes are represented as green, red and black dots, respectively. (B) The dot plot compares changes in nascent-transcript and mRNA expression induced in 1 h-LPS-treated Mo-DCs: results are represented as fold change on a log scale; genes that are induced (>2x), silenced (>2x) and unchanged at the nascent-RNA level are represented as green, red and black dots, respectively. (C) The pie chart shows the percentage of deacetylated genes expressed more than 1 RPKM in immature Mo-DCs. The dot plot shows 1 h-LPS-induced changes in nascent-transcript (red dots) and mRNA (black dots) expression for deacetylated genes; genes are ordered with respect to their change in nascent-transcript expression; positions of representative genes are highlighted. (D) TFBS enrichment analyses were performed for promoters of genes that are induced (>2, 3 or 5-fold), deacetylated and silenced (>2, 3 or 5-fold), or exhibit no change (nc) in expression in Mo-DCs after 1 h of LPS treatment. TFBSs were defined according to JASPAR. The heat map shows the relative enrichment (z-score) of TFBSs that are significantly over-represented (P-value < 10−3). (E) The graphs summarize the z-scores and P-values for all TFBSs in genes that are induced >3-fold (top) or deacetylated and silenced >3-fold (bottom): NF-κB (green) and ETS (red) TFBSs are highlighted.

Mentions:
Examinations of selected genes indicated that nascent-transcript sequencing allows reliable quantification of transcription rates at genes that are silenced or induced in 1 h-LPS-treated Mo-DCs, including protein-coding and microRNA genes (Figure 4B, C and Supplementary Figure S4C). Global analyses indicated that markedly more changes in expression were evident at the primary-transcript level than at that of mRNA-abundance (Figure 5A and Supplementary Table S1). Significantly more genes were down-regulated at the nascent transcript level than at the mRNA level (Figure 5B and Supplementary Table S1), suggesting that most reductions in transcription rate induced by 1 h of LPS stimulation do not yet have a major impact on mRNA abundance. These results demonstrate that assessing gene silencing by nascent-transcript sequencing is significantly more reliable and has strongly improved temporal resolution compared to mRNA-profiling.

Bottom Line:
This silencing response is a rapid primary event distinct from repression mechanisms known to operate at later stages of DC maturation.The repressed genes function in pivotal processes--including antigen-presentation, extracellular signal detection, intracellular signal transduction and lipid-mediator biosynthesis--underscoring the central contribution of the silencing mechanism to rapid reshaping of DC function.Interestingly, promoters of the repressed genes exhibit a surprisingly high frequency of PU.1-occupied sites, suggesting a novel role for this lineage-specific transcription factor in marking genes poised for inducible repression.